Signal transduction pathways evoked by interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) to stimulate expression of other cytokines in mesenchymal cells are not clearly understood. Stimulation of the murine bone marrow stromal cell line +/+-LLDA 11 with IL-1 (500 U/ml) in combination with TNF-α (500 U/ml) (IL-1 plus TNF-α) induced expression of c-jun mRNA as well as granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA. We investigated the possibility that arachidonic acid metabolites, acting through protein kinase C (PKC) and perhaps also through the PKC-responsive transcription factor c-jun/AP-1, may be responsible for regulating GM-CSF transcription in these stromal cells. Expression of GM-CSF mRNA was preceded by IL-1 plus TNF-α induced arachidonate release (assayed using the 3H- derivative). Pretreatment of cells with the phospholipase A2 inhibitor quinacrine (20 μM) inhibited accumulation of both c-jun and GM-CSF mRNA, but had no influence on expression of other genes induced by IL-1 and TNF-α, including leukemia inhibitory factor (LIF). In addition, quinacrine partially blocked IL-1 plus TNF-α induced 3H-arachidonic acid release from prelabeled stromal cells. Furthermore, exogenous arachidonate (10 to 50 μM) induced expression of c-jun. To investigate the role of arachidonate in GM-CSF transcription, we used a reporter vector consisting of the murine GM-CSF promoter linked to firefly luciferase. Transfection efficiency was monitored by assessing expression of a constitutively active gene, RSV-β galactosidase. In this system, quinacrine significantly inhibited IL-1 plus TNF-α induced GM-CSF transcription assayed with the reporter construct. Exogenous arachidonic acid alone (10 μM) increased activity of GM-CSF reporter vector 1.5-fold over control. These results are consistent with the hypothesis that arachidonate metabolites are involved in the signaling pathway that leads to IL-1 plus TNF-α induced GM-CSF gene expression. Thus, transcriptional activation of GM-CSF gene is mediated, in part, by the arachidonate cascade.
|Original language||English (US)|
|Number of pages||8|
|State||Published - Jan 1 1994|
- Arachidonic acid
ASJC Scopus subject areas
- Cancer Research
- Cell Biology